Shen Lanjuan, Ji Cheng, Lin Jian, Yang Hongping
Department of Orthopedics, The First People's Hospital of Hangzhou, No.261, Huansha Road, Shangcheng District, Zhejiang Province, Hangzhou, 310000, China.
Department of Orthopedics, Hangzhou Xiaoshan Hospital of Traditional Chinese Medicine, Zhejiang Province, Hangzhou City, China.
J Bone Miner Metab. 2022 May;40(3):389-401. doi: 10.1007/s00774-021-01308-0. Epub 2022 Mar 25.
A disintegrin and metallopeptidase with thrombospondin type 1 motif 6 (ADAMTS6)-derived circular RNA (circADAMTS6; hsa_circ_0008667) is a novel regulator in interleukin (IL)-1β-induced apoptosis of human chondrocytes (HCs). Here, we planned to probe into its role and mechanism underlying HCs injury in osteoarthritis.
Real time-quantitative PCR and immunoblotting estimated the abundance of RNA and protein, respectively. Cell proliferation and apoptosis were measured by WST-8, EdU, fluorescein isothiocyanate, and caspase3/7 activity assays. Levels of inflammatory cytokines (IL-6 and tumor necrosis factor-α), apoptosis-related proteins (Bcl-2 and Bcl-2-associated X protein), extracellular matrix (ECM)-related proteins (matrix metalloproteinase-13 and collagen type II alpha-1), and PI3K/AKT/mTOR signaling pathway-related proteins (AKT, mTOR, phosphorylated-AKT, and phosphorylated-mTOR) were evaluated by enzyme-linked immunosorbent assays and immunoblotting. Target relationship was confirmed by dual-luciferase reporter, Argonaute-2 immunoprecipitation and RNA pull-down assays.
Abundances of circADAMTS6 and phosphoinositide-3-kinase regulatory subunit 3 (PIK3R3) were underexpressed, and microRNA (miR)-324-5p was elevated in human osteoarthritic tissues and IL-1β-induced HCs. Overexpressing circADAMTS6 and inhibiting miR-324-5p enhanced proliferation and ECM synthesis, but suppressed apoptosis and inflammatory response in IL-1β-challenged HCs. Besides, silencing circADAMTS6 caused similar effects of IL-1β stress on HCs. Mechanically, there was a direct interaction between miR-324-5p and circADAMTS6 or PIK3R3, and IL-1β-induced activation of PI3K/AKT/mTOR signaling pathway was suppressed by circADAMTS6 overexpression and miR-324-5p silencing. Furthermore, counteractive effects of miR-324-5p upregulation on circADAMTS6 overexpression and PIK3R3 knockdown on miR-324-5p silencing were observed.
CircADAMTS6-miR-324-5p-PIK3R3 axis might participate in IL-1β-induced HCs dysfunction via competing endogenous RNA mechanism and the PI3K/AKT/mTOR signaling pathway.
含血小板反应蛋白基序的解聚素样金属蛋白酶6（ADAMTS6）衍生的环状RNA（circADAMTS6；hsa_circ_0008667）是白细胞介素（IL）-1β诱导的人软骨细胞（HCs）凋亡中的一种新型调节因子。在此，我们计划探讨其在骨关节炎中HCs损伤的作用及机制。
实时定量PCR和免疫印迹分别评估RNA和蛋白质的丰度。通过WST-8、EdU、异硫氰酸荧光素和caspase3/7活性测定法检测细胞增殖和凋亡。通过酶联免疫吸附测定法和免疫印迹评估炎性细胞因子（IL-6和肿瘤坏死因子-α）、凋亡相关蛋白（Bcl-2和Bcl-2相关X蛋白）、细胞外基质（ECM）相关蛋白（基质金属蛋白酶-13和II型胶原α-1）以及PI3K/AKT/mTOR信号通路相关蛋白（AKT、mTOR、磷酸化-AKT和磷酸化-mTOR）的水平。通过双荧光素酶报告基因、Argonaute-2免疫沉淀和RNA下拉测定法确认靶标关系。
在人骨关节炎组织和IL-1β诱导的HCs中，circADAMTS6和磷酸肌醇-3-激酶调节亚基3（PIK3R3）的丰度表达不足，而微小RNA（miR）-324-5p升高。过表达circADAMTS6并抑制miR-324-5p可增强IL-1β刺激的HCs的增殖和ECM合成，但抑制其凋亡和炎症反应。此外，沉默circADAMTS6对HCs产生与IL-1β应激类似的影响。机制上，miR-324-5p与circADAMTS6或PIK3R3之间存在直接相互作用，circADAMTS6过表达和miR-324-5p沉默可抑制IL-1β诱导的PI3K/AKT/mTOR信号通路激活。此外，还观察到miR-324-5p上调对circADAMTS6过表达的拮抗作用以及PIK3R3敲低对miR-324-5p沉默的拮抗作用。
CircADAMTS6-miR-324-5p-PIK3R3轴可能通过竞争性内源RNA机制和PI3K/AKT/mTOR信号通路参与IL-1β诱导的HCs功能障碍。
